A mutation in Af4 is predicted to cause cerebellar ataxia and cataracts in the robotic mouse

Abstract

The robotic mouse is an autosomal dominant mutant that arose from a large-scale chemical mutagenesis program. It has a jerky, ataxic gait and develops adult-onset Purkinje cell loss in the cerebellum in a striking region-specific pattern, as well as cataracts. Genetic and physical mapping of the disease locus led to the identification of a missense mutation in a highly conserved region of Af4, a putative transcription factor that has been previously implicated in leukemogenesis. We demonstrate that Af4 is specifically expressed in Purkinje cells, and we hypothesize that the expression of mutant Af4 leads to neurodegeneration. This function was not identified through knock-out studies, highlighting the power of phenotype-driven mutagenesis in the mouse to identify new pathways involved in neurological disease.

Fig. 1.

Progression of Purkinje cell loss in the robotic cerebellum. a–d, Calbindin-stained vermal parasagittal sections at 3 (a), 10 (b), 20 (c), and 42 (d) weeks. Patchy Purkinje cell loss occurs at 10 weeks in the anterior lobes, which is more obvious by 20 weeks of age. At 42 weeks most of the Purkinje cells have been lost, although lobe X is spared, and the anterior lobes are more severely affected. The lobes of the cerebellum indicated (a,I–X) are morphologically unaltered at all time points. Scale bar, 1 mm. e,f, Calbindin-stained transverse sections from control littermate (e) and robotic (f) mice at 20 weeks of age. Parasagittal bands of resistant Purkinje cells in the robotic cerebellum are indicated (arrowheads). Scale bar, 1 mm.

Fig. 2.

Neuropathological findings in the robotic cerebellum. Cresyl violet-stained parasagittal vermal sections at 3 (a) and 42 (b) weeks show shrinking of the robotic cerebellum over time attributable to a decrease in size of the molecular layer, whereas the general morphology of the cerebellar lobules remains intact. Scale bar, 1 mm.c, Calbindin immunostaining shows the Purkinje cell bodies, dendrites, and axons at 5 weeks of age. There is no Purkinje cell loss in robotic mice, but dendrites are thickened (arrowhead) and swellings of the axons are also apparent (arrow). Scale bar, 100 μm. Parvalbumin immunostaining from littermate control (d) and robotic (e) mice shows Purkinje cell loss from the Purkinje cell layer (PCL) at 10 and 20 weeks in parasagittal sections. The decrease in size of the molecular layer (ML) and the resulting increase in cell density are also apparent. Scale bar, 100 μm. f, GFAP immunostaining shows a marked increase in staining density in the robotic cerebellum at 10 weeks of age. The increase occurs in the Bergmann glia of the molecular layer (ML) and in astrocytes in the granule cell layer (GL). Scale bar, 400 μm. g, Ubiquitin immunostaining at 10 weeks of age reveals dot-like deposits in the cerebellum of robotic mice in the granule cell layer (arrowhead) and axon tracts (arrow).

Fig. 3.

Thymocyte development abnormalities in robotic mice. Flow cytometric analysis of littermate control (white bars) and robotic (black bars) thymus at 3 week of age (n = 3). Data for immature T cell markers CD25/CD44 (a) and average SP CD4/CD8 expression (b) are shown as the mean + SEM. Two-tailed Student's t test: *p < 0.05, **p < 0.03, ***p < 0.01.

Fig. 4.

Multiple amino acid sequence alignment of members of the AF4 protein family showing the highly conserved 18 amino acid region within which the robotic mutation lies (arrow). Mouse AF5q31 sequence was taken from an unpublished predicted mouse protein (GenBank accession numberAAG17126). Fugu rubripes sequence was taken from an Ensembl predicted protein of 1123 amino acids, SINFRUP00000087356 (www.ensembl.org/Fugu_rubripes).

Fig. 5.

Expression of Af4 in the cerebellum. Bright-field images of in situ hybridization in littermate control (a) and robotic (b) animals at 5 weeks of age with an antisenseAf4 riboprobe. Expression is confined to the Purkinje cell layer (PCL) in both cases. The molecular layer (ML) and granule cell layer (GL) are marked. The corresponding sense probe is shown (c). Scale bar, 100 μm. d,e, Vermal parasagittal cerebellum sections from littermate control (d) and robotic (e) mice at 5 weeks using the antisenseAf4 riboprobe. Reduced levels of expression are observed in lobe X. Scale bar, 1 mm.